Contents
Introduction ........................................................................................................................................................... 2
Six independent disproof of the sodium pump and the membrane theory:............................................................ 4
The once popular but now abandoned concept of protoplasm............................................................................... 5
The Association-Induction Hypothesis.................................................................................................................. 6
Nano-Protoplasm ............................................................................................................................................... 8
Classic cell physiology: ....................................................................................................................................14
Solute distribution.........................................................................................................................................14
Solute and water permeability ......................................................................................................................18
Cellular electric potential..............................................................................................................................21
Cell swelling and shrinkage..........................................................................................................................27
References ............................................................................................................................................................35
Page 1 of 43
Introduction
The membrane (pump) theory of the living cell taught worldwide today and
used as the foundation for biomedical research is in essence a copy of the one
Theodore Schwann introduced in 1849 in his Cell Theory1,2. Namely, a living
cell is a miniscule puddle of watery solution enclosed in a membrane carrying
pumps. Ideally every educated person should know that this theory is wrong; in
reality few do know.
As a simple starter, I may mention that a typical living cell is solid and
not a water-filled hollow box. Cytologists found this out fast and made
correction promptly 3p4. But so far the great majority of cell physiologists and
biology teachers have not done so. For a clue to this anomaly, I return to the
time when I was a graduate student in the Department of Physiology of the
University of Chicago.
On a spring afternoon in 1947, I was standing on the podium in the main
lecture room of the Department, waiting for my late-coming audience to settle
down. The title of my talk was “The Sodium Pump.”
I began with an apology. I told my audience that even though I had
thoroughly searched the libraries, I had no worthwhile information on the
sodium pump to share. It was just a name. I then went on to other related issues.
When I finally stepped down from the podium, I was startled by what happened
next. Two of my highly respected professors each individually took me aside
and used almost the same words to tell me “ Gilbert, you don’t want to make
yourself a martyr. Leave the sodium pump alone. It is a sacred cow.” I thanked
both warmly for their concern about my personal welfare. At the same time, I
also felt that they overly worried. Next thing you know, I was doing some
simple bread-and-butter experiments to test the validity of the membrane
(pump) theory.
Page 2 of 43
In retrospect, that Schwann should
have presented a wrong view of a typical
living cell is not hard to understand.
With the very limited resolving power of
the early primitive microscope he used
and intent on establishing that cells are
basic units of life, he was naturally
looking for the largest cell that he could
find. What he did find was the large
mature plant cell. As illustrated in Figure
1, each of these mature plant cells
contains a huge central vacuole filled
with clear liquid water. Regarding what
he saw as a larger version of all living
cells big and small, Schwann then
Figure 1 – Mature Plant Cell
elaborated on the structures of the typical
living cell. In specific, he referred to the entire layer of substance surrounding
the body of water as the cell membrane. He also recognized that the chemical
makeup of the fluid in the cell water and the fluid outside the cell was different.
To sustain this asymmetrical solute distribution, he then postulated the
existence in the cell membrane of microscopic devices that regulate the
chemical contents of the fluid inside and outside the cell. Though he did not
explicitly use the word pump, clearly his microscopic devices are what one
would call pumps today. But how and why did the membrane pump become a
sacred cow?
The key to understanding this specific strange event could be in the
“unquestioning acquiescence of the German textbooks” toward what were
introduced in Schwann’s Magnus Opus 4p106. But I have no idea whether or not
that was what my two professors had in mind when they advised me to leave
the sodium pump alone. A safer guess is that they were thinking in general
terms not to do publicly what I did at the departmental seminar.
I did not leave the sodium pump alone because above all, I thought it was
a very serious question that needed to be resolved. I also doubted that anyone
would care whether a lowly graduate student thinks or not thinks. Furthermore,
I had all the tools and frogs to do a test. So I went ahead. I had no idea at the
beginning that my associates and I would one day be able to present the results
of not one, two or even three but six sets of independent experimental studies.
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Six independent disproof of the sodium pump and the membrane
theory:
(1) Energy insufficiency
My early attempt to do some simple study on the validity of the sodium
pump grew into more and more precise inquiries over a period of ten years. The
final and most accurate studies were conducted in September of 1956. Under
rigorously controlled condition, the minimal energy needed to operate the
sodium pump is from 15 to 30 times the maximum available energy if the cell
uses all its available energy for pumping sodium only 5p211;6
(2) Membrane and postulated pump dispensable
Frog sartorius muscle cells with intact cytoplasm but without functional
membrane pumps was given the name, “Effectively Membrane-pump-less
Open-ended Cell” or EMOC preparation. Frog sartorius muscle cells in EMOC
preparations continue to keep intracellular K+ at levels many times higher than
that in the external bathing medium and to keep intracellular Na+ level far below
that in the external medium--- both as found in normal living cells 7p105.
(3) Cytoplasm-free, membrane sac does not work
With both ends tied, exoplasm-free squid axon membrane sacs were filled
with seawater and nutrients and incubated in seawater. This membrane sac
preparation neither pumped K+ into the sacs nor pumped Na+ out of the sacs
against concentration gradients 8p215.
(4)Mobility of intracellular K+ in healthy cells severely reduced
From a total of seventy-two (72) sets of independent studies, the average
mobility of K+ in healthy frog muscle cytoplasm is one eighth (1/8) of the K+
mobility in a dilute salt solution. The K+ mobility rises to 1/2 of that in dilute
salt solution in killed muscles that had kept their non-contracted normal
dimensions. The mobility of K+ in injured cytoplasm falls to between 1/8 and
1/2 of that in dilute solution 9. The validity of an earlier claim that K+ mobility
in squid axons with cut ends is close to that in plain seawater is in doubt on
account of the likelihood of death or severe injury of the axoplasm studied 10p21.
(5) Postulated pump does not have energy to pump Na+ or K+
According to the theory of Nobel Laureate, Skou, an enzyme in the cell
membrane called Na,K- activated ATPase is the sodium pump. The enzyme
Page 4 of 43
was supposed to provide the needed energy for the pumping by splitting and
liberating the energy stored in the high-energy phosphate bond of ATP.
However, Podolsky and Morales have demonstrated clearly that there is no
extra energy to perform work in the mistaken theory of high-energy phosphate
bond 11.
(6) Cell membrane and postulated pump(s) unnecessary to sustain low Na+;
cytoplasm alone does it faultlessly
A mature human red blood cell does not have a nucleus or any other
intracellular organelles. 65% of its weight is water; 34% of its weight is that of
a single pure protein, ferri-hemoglobin. Ling and Ochsenfeld filled sacs made
from narrow dialysis tubing with both ends tied, a 40% solution of pure ferrihemoglobin in water and incubated the filled sacs in solutions containing the
same concentration of 10 mM NaCl but different concentrations of HCl until
equilibrium was reached. Analysis of the equilibrium sac contents revealed a
lowering of the Na+ concentration in the sac water to between 15% to 30% of
the Na+ concentration in the water bathing the sac if its final pH is in the range 2
to 3.. This ultra-simple model (USM) does not have a cell membranes as such
nor the essential constituents of cell membrane and yet it maintains a low
“intracellular” Na+ level precisely matching that found in normal mature human
red blood cells and other living cells. The finding demonstrates that to keep the
low level of Na+ in red blood cells and other living cells, the postulated sodium
pump is at once wrong and superfluous. The cytoplasm alone can do job
perfectly 12. By the way, I will return to this set of study below under the
heading of selective solute accumulation on the significance of the low pH
used.
The mutual agreement among the respective conclusions from these six
independent disproof extends to other evidence not presented here. Since
anyone of these six sets of evidence alone is enough to topple the hypothesis,
together they leave absolutely no doubt that I have not exaggerated the case by
saying that the currently widely taught and used theory of the living cell is
wrong.
The once popular but now abandoned concept of protoplasm
In 1945 French proto-zoologist, Felix Dujardin described a viscous substance
oozing out of crushed protozoa that is glutinous, transparent, insoluble in water
and drawn out like mucus. He gave this living jelly the name sarcode, meaning
fleshy13. Years later, the German plant physiologist Hugo von Mohl also
discovered a similar substance from plant cells and gave it the name protoplasm
Page 5 of 43
14
. It was Robert Remak who suggested using the same name protoplasm for
both sarcode and protoplasm 15 and his suggestion is adopted. After that
landmark episode, two other major historical events occurred in regard to
protoplasm. In 1861,Max Schultze announced his “Protoplasmic Doctrine”
according to which, living cells are membrane-less lump of protoplasm with a
nucleus.” 16. In 1868, Thomas Huxley gave his famous lecture in an Edinburgh
church, in which he referred to protoplasm as the physical basis of life 17.
Generally speaking, the concept of protoplasm remained popular untill at least
the end of the 19th century.
Then the two World Wars came. Among the wreckage was the once
optimistic outlook for the future of life science. More and more began to
believe that the concept of protoplasm is wrong. As an example, the
Encyclopedia Britanica online announced: “ As the cell has become
fractionated into its component parts, protoplasm as a term no longer has
meaning.” My futile search for the word, protoplasm through the indices of the
ten most popular US high school biology textbooks confirmed my worst fear. It
is no longer there.
The Association-Induction Hypothesis
Within the half century since I gave the departmental talk on the Sodium pump,
I have also introduced a unifying theory of living phenomena called the
association-induction (AI) hypothesis 5, put it to extensive experimental testing
and verified it in essence---without any major setback.
Historically speaking, the association-induction model is an heir to the
old protoplasmic concept, which as pointed out above, has been abandoned for
its inability to account for the diversity in color and texture of different
intracellular structures. The resolution of this specific difficulty is one of the
many rewards that the momentous revolutionary change brought to cell
physiology by the AI Hypothesis. In substance, this change echoed what had
happened earlier to physics between mid-18th and early 20th century with the
introduction of the Kinetic Theory of Gases 18. This theory offered the new
concept that gases are not homogeneous matter but are collections of vast
number of small particles or molecules moving randomly in all directions. As
such, the theory explains why gases exert pressure on the container wall in all
directions---a fact totally unexplained until then. It also led eventually to the
long-delayed acceptance by physicists of the reality of atoms (and other
microscopic particles.)
From the standpoint of the four independent revolutionary physicists
involved, it was mostly a lonely and heart-breaking experience. Of the four
Page 6 of 43
who participated, at least one (possibly two) took his own life. And he also
happened to be the great Austrian mathematician-physicist, Ludwig Boltzmann,
who had almost single-handedly given us the major branch of modern physics
called statistical mechanics. One hundred and seventy (170) years elapsed
between the first introduction of the Kinetic Theory of Gases by Daniel
Bernoullli in 1838 and its eventual acceptance in 1908.
For the change brought on by the AI Hypothesis involves the parallel
introduction of microscopic physiology in which molecules, atoms, ions and
electrons replace and give new meanings to macroscopic concepts of
membranes, pumps, rigid pores, semi-permeability etc. Central to this
revolutionary leap under the banner of the AI Hypothesis is the introduction of
the concept of the (unit of) microscopic protoplasm or nano-protoplasm as the
smallest unit of life 19.
Collections of vast number of similar nano-protoplasmic units, in turn,
make up what was once called protoplasm and then discarded. It is now revived
and given the new name, macroscopic protoplasm. Different macroscopic
protoplasm in turn makes up the diverse sub-cellular structures including the
cytoplasm, the cell membrane or the cell nucleus. But macroscopic protoplasm
is not the ultimate physical basis of life just as cells are not the smallest units of
life. Microscopic protoplasm or nano-protoplasm (unit) is the smallest unit of
life as well as life’s ultimate physical basis.
To demonstrate its minute size and simplicity, consider a nanoprotoplasmic unit from the cytoplasm of a mature human red blood cell. It can
be represented by the formula: (Hb)1(H2O)7000(K+)20(ATP)1. Here (Hb)1
indicates the presence in the nano-protoplasmic unit of one molecule of the
protein specific to red blood cells, ferri-hemoglobin or Hb. The other three
symbols in the formula indicate respectively the number of water (7000),
K+(20) and ATP (1) molecule(s) or ions associated with each ferri-hemoglobin
molecule. Assumed spherical in shape, each nano-protoplasm unit measures 8.6
nanometer or 86 angstrom units in diameter.
As a rule, the physiology of the next larger living structure can always be
understood in terms of the then existing knowledge on the physiology of the
living structure one-level or more levels smaller. Accordingly, the vision of the
step-by-step ladder: nano-protoplasm→ macroscopic protoplasm→ organelle
→cell→ organ→individual suggests that the AI Hypothesis is indeed a
comprehensive unifying theory of life that has the potential of explaining all
living phenomena 20.
But for this short summary of the association-induction hypothesis, my
task is first to tell how nano-protoplasm and its component proteins work. The
article will end on the four classic physiological phenomena at the cell and
Page 7 of 43
below cell level: solute distribution, water and solute permeability, cellular
electrical potentials and cell swelling and shrinkage. For AI interpretation of
physiological activities above the cell level, there is a brief summary in
reference 20 p54.
Nano-Protoplasm
The three most abundant components of living cells and their constituents
are water, protein and potassium ion (K+). The individual units of each of these
components in the form of single molecules and single ions are all directly or
indirectly in contact or associated with one another. Through electronic
polarization or induction, each entire nano-protoplasmic unit or their bigger
aggregate can function coherently.
Before presenting the essence of the association-induction hypothesis
proper, I shall briefly describe two subsidiary theories of the AIH. In what is
known as Ling’s fixed charge hypothesis (FCH) first presented in 1952 21, I
pointed out why fixation in space of an ion enhances its association with its
oppositely-charged counter-ion. The intensity of mutual attraction between the
counter-ion and the fixed ion varies directly with the product of their charges
and inversely proportionally to the square of the distance between them.
Although a naked Na+ is smaller than a naked K+, a hydrated K+ is smaller than
a hydrated Na+. Accordingly, a hydrated K+ can stay closer to the singly
charged oxygen atom of an oxyacid group. As a result, preferential electrostatic
adsorption of K+ over Na+ follows. It was pointed out parenthetically that in
muscle cells, the contractile protein, myosin alone carries enough fixed anionic
sites to adsorb all the cell’s K+.
The second subsidiary theory is called the polarized (oriented) multilayer
(PM or POM) theory of cell water first published in 1956 22;23;24. This subsidiary
theory demonstrates why all or nearly all the water molecules in a living cell are
adsorbed as polarized-oriented multilayers on the fully extended protein chains.
When both subsidiary theories of the AIH are considered together, the full
association of all the individual units of all three major components of the nanoprotoplasmic unit is the result.
Page 8 of 43
A nano-protoplasmic unit is an
electronic machine. As such, it can
exist in one of two alternative states:
the resting living state and the active
living state (Figure 2).
Now, the broader subject of life
in general also has two components:
being alive and engaging in life Figure 2 - Resting Living State and Active Living State
activities. Staying in the resting living
state signifies being alive. Engaging in reversible shift between the resting and
active living state represents life activity. Irreversible shift into the active state
ends in the death state.
In the resting living state, nano-protoplasm (or its larger aggregate) is at
equilibrium and therefore does not require continual energy expenditure
for its maintenance. Instead, for a nano-protoplasm unit to assume and sustain
its resting living state, a special region (or site) of the protein component must
be occupied and acted upon by a specific molecular agent called ATP. A
product of the cell’s energy metabolism, ATP serves its functions by its steady
adsorption as such and exerts a far-reaching and powerful electronic impact on
the protein (and its associated molecular and ionic partners.)
In the AIH, both internally produced agents like ATP and external
applied drugs and hormones belong to what are collectively called cardinal
adsorbents 5p118. Cardinal adsorbents fall into two categories: electronwithdrawing cardinal adsorbents or EWC and electron-donating cardinal
adsorbents or EDC. (A third kind, called electron-indifferent cardinal
adsorbents or EIC is of rare theoretical significance and can be ignored.) ATP
and calcium ion (Ca++), for example, are highly important EWC’s. To explain
why and how the binding of a cardinal adsorbent like ATP achieves its farreaching physiological impacts, we need some additional knowledge on the
structure and function of proteins and other chemicals..
Among the three major components of living cells, protein alone is made
only by living cells of one kind or another and thus unique to life. As a rule, the
proteins in different kinds of nano-protoplasm are different. As a long-chain
polymer, the building blocks of a protein are some 20 different small molecules
called α-amino acids or simply amino acids. Each amino acid can be
represented by the general formula, NH2CHRiCOOH, where Ri differs among
Page 9 of 43
different amino acids but the remainders are almost always the same. The
symbol RI for the amino acid, glycine, is a simple neutral H atom, that for
alanine is a neutral methyl group, that for aspartic acid is COOHCH2 with a
negatively charged, anionic β-carboxyl group hanging on its end. For the amino
acid, glutamic acid, it is COOH(CH2)2 with an anionic γ-carboxyl group at its
end. For the amino acid lysine, it is a NH2C(H)2(CH2)3 with a terminal
positively-charged cationic ε-amino group. For arginine, it is (NH2)2H(CH2)3
with a terminal cationic guanidyl group.
Equation 1 below shows how a miniature protein or short polypeptide or
tripeptide containing two peptide linkages (CONH) is formed by joining three
free amino acids with the loss of four hydrogen atoms and two oxygen atoms in
the form of two water molecules.
NH2CHR1COOH + NH2CHR2COOH + NH2CHR3COOH ↔
NH2CHR1CONHCHR2CONHCHR3COOH + 2 H2O
(1)
As a part of a protein molecule, what remains in the polypeptide or protein of
each amino acid is called an amino acid residue. The symbol RI that each amino
acid carries is called a side chain. In a neutral medium, the β- and γ-carboxyl
groups are fully ionized and thus each endows the protein with a single negative
electric charge of a fixed mono-valent anion. Each ε-amno group and guanidyl
group, on the other hand, endows the protein with a positive charge in the form
of a fixed mono-valent cation. β- and γ-carboxyl group may function as
adsorption sites for K+, Na+ or fixed cations. Pairing of a fixed cation and a
fixed anion forms a salt linkage 25. The peptide linkage, CONH, may bind onto
similar peptide group on the same protein chain fourth removed in either
direction, forming an α-helix structure or alternatively bind multilayers of water
molecules. The choice between the two is determined by the electron density of
the sites involved, a subject which the section farther below will discuss.
Above all, alternative structure of the peptide linkage undergoes
extremely rapid transition or resonance 26, which makes the polypeptide chain
highly polarizable and thus suitable for long-distance information and energy
transfer. In addition, resonance also endows short-range negative electric charge
to the carbonyl oxygen atom and positive charge to the imino NH group. That
fact too is of great importance.
The c-value and the c-value analogue and their respective role in
determining selective K+ and water adsorption.
Page 10 of 43
As mentioned already, in a neutral medium like that provided by the
tissue fluid, blood plasma or Ringer’s solution, the β-, and γ-carboxyl groups
are fully ionized because their respective acid dissociation constants
Ka’s are high enough. Another modified quantitative parameter is the pKa; it is
the negative logarithm of Ka to the base 10. As an illustration, the pKa of
vinegar or acetic acid is 4.76 while the trichloroacetic acid (TCA) is so much
stronger acid that its pK a is below unity. The cause for this difference is the
classic induction mechanism. Now, each chlorine atoms has 17 positively
charged protons in its nucleus whereas a hydrogen atom has only one proton in
its nucleus. Each proton carries a net positive electric charge. Thus replacing
the three H atoms in the acetic acid CH3COOH with three chlorine atoms to
produce a TCA, also generates a far-reaching inductive effect so that the
electron density of distant singly-charged oxygen atom of the carboxyl group
COO- becomes greatly reduced. As a result, a sharp fall of its pKa occurs as just
pointed out above.
However, pKa is not an independent parameter, it is limited to interaction
with a specific partner, the H+ ion. For our broader inquiry, I introduced as part
of the association-induction hypothesis, the independent parameter called the cvalue. Rigorously defined elsewhere 5p58;10p126, it is in units of angstroms and it
can be regarded as a measure of the electron density of the singly charged
oxygen atom of an oxy-acid group. A high c-value is equivalent to a high pKa.
A low c-value is equivalent to a low pKa.
With the c-value defined, I was able to compute the theoretical binding
energies of K+ , Na+ , Rb+, Cs+. Li+ as well as those of
H+ and NH4 on fixed anions with different c-values
5pp62to78
Figure 3 represents one set of the
.
theoretically computed results, which shows that for
any pair of ions like K+ and Na+, there is a cross-over
point at a certain c-value. In other words, if the cvalue of a group of β-, or γ-carboxyl groups is kept
low, they would have a selective preference for
adsorbing K+ over Na+. On the other hand, if the cvalue of the same β-, or γ-carboxyl groups is made to
rise to a higher c-value, they may change to
preferring Na+ over K+. In addition, I also introduced
a c’-value, which represents the positive charge
density of fixed cations like an ε-amino group of a
lysine residue.
The binding or removal of cardinal adsorbent
Page 11 of 43
Figure 3
like ATP is the main controlling events. However, under what is called
“Principle of Additivity” 5p95, the AI Hypothesis also suggests that anything that
binds onto the protein component of a nano-protoplasm exercises an electronic
impact, though the polarity and magnitude vary from one adsorbent to another.
An in-depth examination of the amino acid composition of many proteins
led to the conclusion that physiologically active proteins as a rule contain a high
percentage of aspartic and glutamic acid residues. As a result these proteins
contain a high percentage of β-, or γ-carboxyl groups. For reasons that would be
made clear in sections to come, one may say that these carboxyl groups on
relatively short side chains function as the eyes and hands of the smallest unit of
life, nano-protoplasm. Another type of important reactive functional groups of
cell proteins is the carbonyl groups (CO) of the peptide linkages (CONH). The
peptide linkage is bipolar. Seen from a distance, the group as a whole is
electrically neutral. However, as one approaches the group closer and closer
from different angles, the individual carbonyl oxygen atom becomes a fixed
anion and the imino NH becomes a fixed cation. For the counterparts of the cvalue and c’-value, I also introduced for the carbonyl oxygen and for the imino
group the independent quantitative parameter c-value analogue and c’-value
analogue respectively 5p57.
As a part of the AI Hypothesis,
I have shown that a list of each amino
acid’s empirically-determined relative
tendency to form α-helical structure
called the α-helical potential is
strongly correlated with the electron
donating strength of the side chain of
each amino acid (Table 1) 27;28;29.
In consequence, the carbonyl
oxygen with high c-value analogue
has a high tendency to form α-helix,
driving the protein to form a folded
secondary structure. As its alternative,
carbonyl oxygen atoms with a low cTable 1
value analogue prefer to stay open,
polarizing and orienting multilayers of adsorbed water molecules.
Physiological reversible degradation of ATP drives the cell into the
active living state of a life activity. On the other hand, with the key controlling
agent ATP in place, individual molecules or ions of the bulk of all three major
Page 12 of 43
components are directly or indirectly in contact with one another and function
coherently together. This “connectedness” is achieved through a combination of
both short-range inductive and (falling-domino-like) long-range information
and energy transmission mechanisms. Basically they are versions of electronic
polarization of one kind or another. Substitution of one covalently linked
chemical group by another covalently linked group produces in theory two
kinds of impacts. An effect mediated through the intervening space is
traditionally called a Direct effect or D-effect; the impact on a target group
mediated through intervening linked atoms is called an Inductive or I effect 30.
Combined D and I effect is called an F-effect 31.
The classic Induction theory introduced by G. N.
30
Lewis is limited to changes in covalently linked atoms or
radicals. In developing the AI Hypothesis, I demonstrated
that both the initiator groups and target groups of an
inductive effect can be linked to the protein involved by ionic
or H-bonds 10pp113to115. In addition, distance action was sorted
out into two kinds: Short-range static effect is called Direct Feffect and long-range dynamic effect is referred to as Indirect
F-effect. In the year 2008 I gave the Indirect F-effect a new
name, the AI cascade mechanism, where the letter A and I
stand for association and induction respectively 19p137.
We now know that the Direct F-effect (mostly
inductive) can be transmitted through three peptide linkages
with or without an additional small stretch of saturated
Figure 4
carbon chain 32pp47to50. Like a chain of frictionless tethered
seesaws, the AI cascade mechanism in theory can go on as
far as it takes to reach the end of the protein molecule. The Direct F-effect is
simple and readily understood. In contrast, the AI cascade mechanism as
illustrated in Figure 4 is more complex as described in ref. 10 pp 147-149 and in ref.
20 pp 90-95. In broad terms, the AI cascade allows a cardinal adsorbent to achieve
three mutually complimentary goals: (1) from here
to there; (2) from one to many; (3) make many
behave like one. Indeed, in Figure 5, the
quantitative correspondence between the theoretical
predicted patterns relationships as shown in A and
experimental observations shown in B, C, D and E
Figure 5
demonstrate the power of the combined actions of
the short-range Direct F-effect and long range AI cascade mechanism 33p176.
Page 13 of 43
Now that we have completed the presentation on how the ultimate unit of
life, nano-protoplasm works, we are ready to present the AI interpretation of the
four classic cell physiological subjects namelysolute distribution, water and
solute permeability, cellular electrical potentials and cell swelling and
shrinkage
Classic cell physiology:
Solute distribution
The main subject in Figure 11.21 of ref. 79 shows the equilibrium K+ contents
of frog muscle in Ringer solution with different K+ concentrations but a fixed
Na+ concentration of 100 mM. In outline, the curve bears strong resemblance
to that of oxygen uptake in intact human red blood cells shown in the inset.
Both curves are S-shaped 34p346. In the case of oxygen uptake, the conventional
view is that the oxygen is taken up by binding onto the cell protein hemoglobin.
As such, it indicates that as the external oxygen concentration rises steadily,
the heme site with weak binding energy for oxygen takes up oxygen first,
followed by uptake by heme sites with stronger binding energy.To explain this
anomaly, the “Crevice theory” was suggested. In this concept, the heme sites
with strong binding energy are buried inside “crevices” of the hemoglobin
molecule while the heme sites with weak binding energy are outside the crevice
and thus can bind oxygen first. Perutz’s precise work on the structure of the
hemoglobin molecule revealed that all 4 oxygen-binding heme sites are on the
protein molecule and none buried, leaving the prominent S-shaped curve unexplained. This shortcoming was resolved in 1964 with the introduction of what
has become known as the (auto-cooperative) “Yang-Ling adsorption
isotherm” 35;10pp137 to 140.
To show how useful this adsorption isotherm could be, I have plotted in
Figure 6 the theoretical curve to match the unusually
accurate experimental data of Lyster carried out in the
laboratory of Prof. F.J. W. Roughton of Cambridge
University 36. All 16 data points fall on or very close to
the theoretical curve. And there are just two variables in
the theoretical curve: the intrinsic equilibrium binding
constant, Kooj→i and the nearest neighbor interaction
energy, –γ/2, as the energy gain each time a new ij pair
is formed on two adjacent sites. Similarly, it was shown
that the K+ and Na+ equilibrium distribution also
follows the dictate of the Yang-Ling auto-cooperative
Figure 6
Page 14 of 43
isotherm and as such also described by just two constants of similar
magnitudes. Oxygen binds onto the 4 heme sites on hemoglobin; K+ and Na+
bind onto many more β-, and γ-carboxyl groups. Yet, the heme sites we refer to
as closest neighbors are in fact far apart-- separated by different number and
kind of amino acid residues. Similarly, the nearest- neighboring β-, and γcarboxyl groups are also far apart and separated by different number of amno
acid residues. The AI hypothesis can readily explain these because the AIcascade mechanism makes a long chain of peptide-to-peptide linkages as if they
were one. That is, like the energy of one falling domino, it is the same as the
next one. In the AI Hypothesis, cooperative changes like those just described
belongs to the category of spontaneous cooperative transition (Figure 7A) in
contrast to the category of controlled cooperative transition, (Figure 7B) a
subject we will deal with next.
Figure 7
Since we do not have the technology to study a single nano-protoplasmic unit,
mature human red blood cells are a special gift to investigators like myself
because they contain virtually just a single kind of protein, ferri-hemoglobin
and mature human red blood cells are also easy to obtain anywhere. We have
already used mature human red blood cells to illustrate the small size and
simplicity of a nano-protoplasm unit and Lyster had unwittingly demonstrated
the accuracy of the Yang-Ling cooperative adsorption isotherm.
Another gift to the like of myself is the frog muscle. A frog muscle cell is a
more complex cell than a human red blood cell. Indeed, what we often study is
usually not a single cell but a collection of similar (and some dissimilar) cells
belonging to a single whole muscle organ. And yet as a rule, it too behaves like
mature human red blood cells, thereby demonstrating that physiologically
active cells share proteins that are not unlike hemoglobin.
Page 15 of 43
Figure 8 shows what happened to a collection of frog muscles when
exposed to a low concentration of the metabolic poison, sodium iodoacetate and
radioactively labeled
sucrose. Notice that as
the ATP concentration
fell, its time course of
decline runs parallel to
that of the falling
concentration of K+ but
anti-paralleled to those
of the rising curves of
both Na+ and sucrose.
For solutes like Na+
and sucrose, which
exist inside the cell at
concentration
below
Figure 8
their
respective
concentration in the
bathing solution, the extra intracellular concentration gained could be either
adsorbed or free.
We have previously demonstrated that sucrose shows strictly rectilinear
distribution in frog muscle 34, indicating a lack of significant binding in muscle
cells. With this in mind, we reached the conclusion that the parallel rising time
courses of labeled sucrose and Na+ reflect a steady desorption of the bulk phase
cell water and steady rise of their respective q-values. This, in turn, affirms that
the ATP concentration determines the state of polarization-orientation of the
cytoplasmic nano-protoplasmic water--- like that illustrated in Figure 2. Since
the backbone carbonyl groups adsorb water in multilayers at low c-value
analogue, the data adds yet another set of data affirming that ATP is an
electron-withdrawing cardinal adsorbent or EWC. In the next section, I present
results of a parallel study on K+ and Na+ distribution in similar frog muscles
under the influence of an EDC.
To avoid inadvertently perturbing the ATP level of the frog muscle at the
same time, we added an extremely low concentration of an electron-donating
cardinal adsorbent (EDC) a cardiac drug called ouabain 37 to the bathing
solution containing an increasing ratios of the concentration of K+ over Na+. All
the data points in Figure 9 are experimental and the pairs of X-shaped curves
are once more theoretical according to the Yang-Ling isotherm. What the drug
Page 16 of 43
did was to reduce the value of Kooj→I from 100 to 21.7. The result shows that at
the physiological concentration ratio of 2.5 x 10-2, the entire population of
β-, and γ-carboxyl groups changed its adsorption partners from all K+ to all Na+.
Figure 9
In yet another similar low-ouabain level experiment 38, we assumed that
the muscles’s cardinal sites took up all the added ouabain added to the bathing
solution. We then showed that each one of the adsorbed ouabain molecule
would have caused one thousands and forty two (1042) β-, and γ-carboxyl
groups to switch their adsorption partners from all K+ to all Na+. In still another
parallel analysis of the far reaching impact of ATP, we showed that at least
8000 water molecules are under the influence of one adsorbed ATP
molecule20p40.. Both sets of number illustrate and confirm the far-reaching
influence mediated by the AI cascade mechanism.
Lastly, I return to the study already described as the 6th disproving the
memebrane pump hypothesis 12. In that study I pointed out how according to the
AI Hypothesis of Na+.partial exclusion from cell water began with a higher
water-to-water interaction of cell water. It follows that more energy required to
install a hydrated Na+ in the cell water than that recovered in filling up the hole
left behind in normal liquid water. And the water-to-water interaction energy is
higher because all the cell water is adsorbed as polarized-oriented multilayers
on the exposed polypeptide chains of cell proteins. The existence of cell
proteins in the fully extended state in turn is in consequence of the adsorption
on key cardinal sites of the protein of ATP, which is an electron withdrawing
cardinal adsorbent or EWC. And in the super-simple model or (SSM) studied
we did not include ATP as such, which is only assumed to be an EWC. Instead,
we replaced it with an artificial EWC, the H+, which being nothing but a
positive electric charge, is thus by definition an EWC . The fact that our
assemblies of artificial human red blood cell cytoplasmic nano-protoplasm units
did reproduce precisely low Na+ level as seen in normal red blood and other
Page 17 of 43
cells affirms not only the AI hypothesis of solute distribution, it also added
another set of convincing evidence that ATP is an EWC.
Solute and water permeability
Radioactive elements used as tracers in biological studies became available in
the wake of WWII. For the first time in history, biologists could measure the
genuine permeability of cell membranes to solutes like Na+ for example. Soon
the historic macrosopic concept that the normal cell membrane is absolutely and
permanently impermeable to Na+ was unequivocally disproved. It was in the
crisis thus created for the suvival of the membrane theory, that the membrane
pump concept introduced by Theodor Schwann was brought center stage again.
Ironically, it was with the deployment of similar radioactive Na+ tracer that the
sodium pump idea was put away for good. But that was only the beginning of
adventures brought on by the radioactive tracer technology in cell physiology.
The most abundant component of nano-protoplasm is water. According
to the AI Hypothesis, each water molecule is linked to the protein directly or
indirectly as a part of polarized-oriented multilayers of water molecules.
Furthermore, as a general principle, the nano-protoplasm making up organelles
like the cell membrane should not be profoundly different from nanoprotoplasm making up the bulk-phase cytoplasm. Two iconoclastic predictions
would follow as a result. First, the traditional belief that the cell membrane is
primarily a continuous (phospho) lipid bimolecular layer is wrong. Second, the
rate of diffusion of labeled water molecules could be similar in their passage
through the cell membrane as in their passage through the bulk-phase
cytoplasm. In the terminology of diffusion lingo, this type of diffusion is a bulkphase limited diffusion in contrast to a surface limited diffusion. With the help
of advanced mathematics of diffusion on hand, it was not difficult for me to
introduce what is called an influx profile analysis to detect and find out which is
which 39. An additional requirement for making accurate studies of bulk-phaseor surface limited diffusion is very large cells. Those we chose are the frog
ovarain eggs and the giant muscle cells of giant barnacles.A third critical
requirement is radioactively labeled water. This was available in the shape of
tritiated water that can be measured in a β-counter.
Thus equipped, my associates and I carried out two main projects: one on
frog ovarian eggs by Ling, Ochsenfeld and Karreman 39 and the other on giant
barnacle muscle fibers by Reisin and Ling 40. The results were unequivocal; the
diffusion of labeled water in both of these giant cells was bulk-phase limited.
Page 18 of 43
A by-product of this study is the diffusion coefficient of labeled water
throughout the inside of the two types of giant cells. It is equal to 30% to 60%
of that of normal liquid water in the frog ovarian eggs and 55% of that of
normal liquid water in the giant barnacle muscle fibers. Furthermore, these
figures agree perfectly well with figures from four groups of other independent
investigators of the giant barnacle muscle fibers, using different experimental
methods.of study. Thus, in units of 10-5cm/sec, their results are 1,34 (Caillé and
Hinke), 1.56 (Abetasedarskaya et al), 1.20 (Finch et al) 1.35 (Walter and
Hope), averaging 1.36 ±0.13 (s.d.). From eight sets of studies, our result are
1.35 ± 0.125 (s.d.) 19p190. This pair of investigations produced yet another set of
major confirmation of the AI Hypothesis and refutation of the membrane
(pump) theory.
Page 19 of 43
This finding also provides an explanation
why (the failed) antibiotic, valinomycin increases
a thousand-fold the permeability of K+ through
pure phospholipid membranes, have no influence
at all on the K+ permeability of frog ovarian eggs,
frog muscle, human lymphocytes, inner membrane
of rat liver mitochondria (Figure 10) 41.
Valinomycin has no detectable impact on the their
K+ permeability because the dominant continuous
phase of the cell membrane of these cells are
polarized-oriented
water
molecules,
not
phospholipid bilayers.
Figure 10
Next I shall deal with the membrane permeability toward vital nutrients
like D-glucose that is carried in the blood stream. The problem raises the
question of extreme diversity, which is lipid membrane theory does not
provide.Thus to reach the cell interior of the functional cells in need of that
specific nutrient, D-glucose must be able to pass through speedily the
membrane of the blood capillary wall in addition to the membrane of the
recipient cells. For that, the requirement is quick permeation and hence high
membrane permeability 42. In contrast, for animals like the frog which spend its
life in large body of pond water, the outer skin must have low permeability
toward D-glucose or else they will all be lost to the pond water 43. Again the
AI Hypothesis provides a reasonable answer of this striking divergence: in
theory the degree of polarization and orientation of the rate-liming water
multilayers in the cell membrane are not fixed but flexible.
Thus, the excess water-to-water interaction energy in water assuming the
state of polarized-oriented multilayer is a very small figure (126 cal for bulk
phase frog muscle cytoplasm), which is a minute number when compared to the
total water-to-water interaction energy or vaporization energy of water (10,000
cal.) Thus in theory, a doubling the tiny flexible number 126 cal could greatly
slow down the permeability toward large solutes like D-glucose while halving it
would greatly increase its permeability. Yet doubling or halving a minute
number produces no more than two more minute numbers and as such readily
achievable. This theoretical flexibility is well affirmed by experimentallydetermined extremely high permeability toward large solute like D-glucose in
normal frog muscle cell membrane and orders of magnitude lower permeability
toward nutrients like D-glucose in membranes of outer frog skin.
Page 20 of 43
High permeability to glucose, even
sucrose is shown in Figure 11 and
exceedingly low permeability of the
inverted frog skin toward L-glucose
and sucrose as shown in Figure 12.
Figure 11
Figure 12
In contrast, without the fundament flexible provided by continuous phase
of polarized water, lipid membrane models like that studied by Collander would
make all cell membranes even more impermeable to glucose than the inverted
frog skin10p207.No cells with that kind of membrane could survive or indeed
exist.
Cellular electric potential
In mid-19th century, Emile DuBois-Reymond, a student of Johannes Müller at
the Berlin University and a member of the “Reductionist Four”, introduced the
name, demarcation or injury potential for the standing electric potential
between the injured end of a muscle or nerve and its intact surface. He also
gave the name, action potential to the negative Schwankung (negative variation)
that propagates along the length of a nerve or muscle fiber. A student of another
member of the “Reductionist Four”, Hermann von Helmholtz was Julius
Berstein. Bernstein was in his sixties when he introduced in 1902 his membrane
Page 21 of 43
theory of cellular electrical potentials. In this theory, a semi-permeable cell
membrane bars the passage of Na+ and all anions, thereby converting what
would be a transient diffusion potential difference into a lasting one. Bernstein
also suggested the theory that during the passage of an action potential, the cell
membrane becomes momentarily permeable to all ions. Subsequent study of
giant squid axons, however, revealed that the action potential does not represent
a mere transient annulment of the inside-negative membrane potential but
involves a transient polarity reversal creating the an inside positive “overshoot”
44
.
When I began my carrier as a graduate student under Prof. Ralph W.
Gerard of the Department of Physiology of the University of Chicago, my
immediate task was to improve the capillary glass electrode used to measure the
membrane potential of individual impaled frog muscle cells or fibers. Success
in my early efforts of perfecting the production and use of the microelectrode
permitted us to make accurate measurements of the membrane potential of frog
muscle cells. (And in time to come, also measuring a great variety of big and
small living cells and intracellular organelles by other investigators.) My early
work confirmed the proportionality of the resting potential to absolute
temperature and to the logarithm of the external K+ concentration.
Notwithstanding, the unequivocal demonstration of membrane permeability to
Na+ threw a monkey wrench in the Bernstein’s membrane theory, making the
theory as such no longer tenable.
In 1949 Hodgkin and Katz made a historically great discovery 45.
Namely, the increase of membrane permeability during the passage of an action
potential does not involve an increase in the permeability toward all ions but is
restricted to that of one ion, Na+. Furthermore, the height of the overshoot is
directly proportional to the logarithm of the external Na+ concentration. In face
of the by-then widely recognized permeability of the cell membrane to
Na+, Hodgekin and Katz then introduced the so-called Hodgkin-Katz-Goldman
equation for the cellular electrical potential, :
 = RT/F{ ln ( PK[K+]in + PNa [Na+]in + PCl[Clin- ) / (PK[K+]ex + PNa [Na+]ex +
PCl[Cl-]ex) },
(2)
where R and T are the gas constant and absolute temperature respectively. F is
the Faraday constant. PK , PNa , PCl are the permeability constant for the three
ions respectively and [K+]in and [K+]ex are the intracellular and extracellular K+
concentrations, etc.45. Then, just as the demonstrated permeability to Na+
wrecked Berstein’s theory of membrane potentials, demonstrated indifference
Page 22 of 43
of the cellular resting potential toward external concentration of the highly
permeant Cl- 46 made untenable the Hodgkin-Katz-Goldman equation.
In this tumultuous time, I received my Ph.D. degree in physiology. The
year was 1948. My Ph.D. thesis 47 as well as my first four major papers
published conjointly with Prof. Gerard and Walter Woodbury all bore the name,
membrane potential in their respective titles 48. Since all these are part of the
membrane (pump) theory, which I began to suspect to be wrong. I began to
think of rejecting Bernstein’s membrane potential theory and constructing an
alternative theory of the cellular resting and action potential. But at that time, all
I knew was what I had been using as a theoretical guideline is wrong. But I had
no idea whatsoever what to replace it with until three years later.
Then on a fine day in 1951, a new idea suddenly dawned on me that
would provide a new way of selectively accumulating K+ over Na+ without
continual energy expenditure. Announced briefly in 1951, it was published in a
full-length article in 1952 and would be referred to henceforth as Ling’s Fixed
Charge Hypothesis21.
Shortly after the publication of Ling’s Fixed Charge Hypothesis, I
announced in 1955 and again later, at the Federation Meeting at Atlantic City
my new belief that the resting potential of living cells share a fundamental
mechanism with that of the glass electrode 49;50;51. The name eventually given
many years later to this new model is Closest contact surface adsorption
potential (CCSA) 33p.206;p226.Two simple equations were first introduced for what
I will henceforth refer to as the non-committal resting potential:
 = RT/F{ln ( KK[K+]in + KNa [Na+]in )},
(3)
and
 = RT/F{ln (1 + KK[K+]in + KNa [Na+]in )},
(4)
where KK and KNa are the adsorption constants for K+ and Na+ on the surface of
the living cells.
The change from the membrane potential model to the close-contact
surface adsorption model in fact represented a shift from a macroscopic model
of membranes and semi-permeability to a microscopic model of ion adsorption
on and desorption from β-, and γ-carboxyl groups at the cell surface.
Historically speaking, this change has a rich and interesting background.
In 1881, Ludwig Helmholtz measured the electric potential difference
across two electrolyte solutions separated by a thin glass membrane 52. At that
time, it was believed that the glass membrane is permeable to H+ and
Page 23 of 43
impermeable to other ions. Max Cremer, professor of physiology at the Berlin
University, suggested for the first time in history that the glass membrane is a
suitable model for the living cell membrane 53. Then something extraordinary
happened. A scientist by the name, Horovitz alias Lark-Horovitz showed that a
regular glass membrane electrode with no permeability toward the silver ion
(Ag+) acquired a sensitivity to this ion after soaking in a silver nitrate solution
overnight 54. Horovitz attributed the electrical potential difference to originate
from the presence of negative electric charges on the surface of the glass
electrode and accordingly called this potential a surface adsorption potential.
But this concept was not new even then.
In 1892, physicist W.H. Nernst first suggested that the Law of
Macroscopic Electro-neutrality forbids the movement of significant electric
charges within each phase. It was only at the phase boundary, that ions
accumulate and generate electric potential differences 55. Baur and Kronmann
demonstrated the addition of strychnine to one electrolyte solution separated
from another by an oil layer, generates what they call surface adsorption
potential 56;57. However, glass membrane and oil layer are not the only materials
used to construct inanimate models of cell electric potentials. A third kind is
made of collodion.
Nitrocellulose also known as gun cotton is made by exposing cellulose to
concentrated nitric acid. In the commercially available form called collodion,
nitrocellulose is dissolved in a mixture of alcohol and ether. Dipping a glass
tube in collodion, followed by drying in a humidified atmosphere, a “thimble”
of membrane electrode could be slipped off for studying membrane potentials.
In response to Horovitz’s startling conclusion on the key role of fixed electric
charges, Leoner Michaelis and W. Perlzweig pointed out that their colloidion
thimble electrode does not carry fixed charges on its surface and therefore
continues to affirm Bernstein’s theory of membrane potentials 58.
Then World War II broke out. The German chemical company could no
longer provide Michaelis’s laboratory with more collodion to make their
collodion membrane models. In response, Michaelis’s students began to make
their own colloidion. To their disbelief, the purer the collodion they made, the
worse they became as a model for the membrane potential. The models made
from the perfectly pure collodion, showed no electric potentials at all.
Eventually, the cause of this mystery was discovered. It turned out to be
an impurity in the Schering collodion that generated the electric potential. What
is that impurity? It turned out to be our familiar carboxyl groups 59. This strange
turn of events again demonstrated that it is the microscopic surface carboxyl
groups that give rise to the potential. It is not a macroscopic potential across a
semi-permeable membrane.
Page 24 of 43
But oil membrande continues to yield interesting knowledge. Thus
Collacico tried to measure the electrical potential difference between two
potassium chloride (KCl) solutions of different strength separated by a layer of
oil. But he found none. However, when he added some anionic detergent like
sodium dodecyl sulfate to one KC l solution, then the oil surface facing that
solution becomes a cation-electrode and as such sensitive to the K+
concentration in that compartment but totally indifferent to the K+ concentration
in the other compartment. On the other hand, if instead of the anionic detergent,
sodium dodecyl suface, he added a cationic detergent like
cetyltrimethyammonium bromide then the surface of oil layer facing this KCl
solution becomes an anion electrode sensitive to the Cl- concentration. Now it is
no longer sensitive to the K+ concentration in that compartment 60. This study
was further confirmed and extended by Tamagawa and Nogata 61. All in all, the
Colacicco study dramatically affirmed the microscopic surface adsorption
model like that in the CCSA model to come later.
Next, I shall describe how my associate and I combined the two failed
model (glass membrane model and collodion membrane model) into a
spectacularly successful model called (oxidized) colloidon-coated glass (CCG)
electrode 62. The procedure of preparation is to coat a Corning O15 glass
electrode with a layer of collodion, allowed it to dry in a humidity chamber. A
brief exposure to a NaOH solution followed, causing oxidation of the
nitrocellulose to form more carboxyl groups on the electrode’s surface.
It is almost unbelievable how closely this model behaves electrically like
a living frog muscle cell: (1) both show a relative sensitivity toward the alkalimetal ions in the order Rb+> K+ , Cs+> Na+ (2) both are totally insensitive
toward Mg++, (3) both are two orders of magnitude more sensitivity toward H+
than to K+.
In explanation, I point out that cellulose like protein can also exist in a
folded and extended conformation. The carboxyl groups appear to resemble
living cells in having a low c-value. This too is understandable because the nitro
group added onto the cellulose is a well-known electron-withdrawing
radical5p162fig47. As such its incorporation took the place of the binding of the
EWC ATP in the living frog muscle.
The action potential
According to the theory widely taught in textbooks, the action potential
involves the sequential opening of specific sodium gates and potassium gates.
Driven by an alleged sodium potential, Na+ from the external medium rush into
Page 25 of 43
the cell, creating the overshoot. Subsequent opening of the potassium gates
allows K+ to leave the cell creating the after potentials.
Like the long-disproved membrane potential, this widely taught action
potential model is also untenable. First, the assumption of the existence of
standing “Na+ potential” is unrealistic because the resting cell membrane is
highly permeable to Na+. And if such a “Na+ potential” existed, it would
promptly be discharged 33p225. Similarly, it has been established that the alleged
sodium gates also permit the passage of K+ 10p303p310;33p226. That means when you
open the sodium gate, not only would external sodium ion rushing into the cell
interior, intracellular K+ would at the same time rushing outward also, therefore
annulling the potential change due to Na+. But above all, the disproof of the
membrane (pump) hypothesis makes it superfluous to consider these details.
They are presented to demonstrate that local issues also disagree with the
widely taught models.
The AI Hypothesis of the action potential stands on the solid foundation
of the model of the living cell in general and the resting potential in specific as
described earlier. There is another outstanding feature of the muscle, nerve and
other excitable cell surface involved: Unlike the cell interior, these fixed anions
are not accompanied by an equal number of fixed cations as in the nanoprotoplasmic unit shown in Figure 2. Following the Faraday Cage Effect, their
surface layer is primarily
anionic.
Another
distinguishing feature of
the excitable cell surface
is
the
controlling
influence
of
the
++ 64
(auxillary) EWC, Ca
.
Local
electrical
perturbation created by
the advancing front end
of a coming action
potential
or
the
depolarization
of
end-plate
potential
5pp358to380
++
causes the detachment of the Ca (Figure
13).
Figure 2
The ensuing action potential is an expression of
the surface macroscopic protoplasm made of vast
number of nano-protoplasmic units like that
Page 26 of 43
Figure 13
shown in Figure 2 from right to light. The main event is the description of the
adsorbed-oriented multilayer of cell surface water. This liberation of water
molecules instantly creates a transient sodium potential and with it, Na + from
the plasma or Ringer’s solution rushes into the cell producing the “overshoot”.
The K+ liberated during the auto-cooperative shift as well as the K+ displaced by
the inflowing Na+ give rise to the action potential.
A critical support of the
AI model of the action
potential was provided by the
work of Villegas et al 65 and
shown in Table 2. The data
obtained showed that repeated
Table 2
stimulation of giant squid
axons increase the permeability of the squid axon surface to erythritol, mannitol
and sucrose, which are all found only in the cell water, none adsorbed. Their
increased entry during the passage of the action potentials is thus the likely
consequence of the transient depolarization and liberation of the nanoprotoplasmic water. In farther support of this belief, the increase of these nonelectrolytes uptake will not take place in the absence of Na+ in the bathing
solution.
Cell swelling and shrinkage
Abbé Nollet was a science teacher of King Louis XV of France. An opponent of
Benjamin Franklin’s “One fluid theory of electricity”, Nollet was also the first
recorded investigator of the osmotic phenomena generated by a semi-permeable
membrane 66. To begin, Nollet filled a bottle with alcohol. He then covered the
mouth of the bottle with a flattened piece of pig’s bladder and tied it down
firmly with a piece of string before immersing the bottle in a tub of water.
Hours later, he found that the bladder membrane bulged outward, indicating
more fluid had gone into the bottle than out of the bottle. On the other hand, if
he filled the bottle with water and immersed the bottle in alcohol, the membrane
would bulge inward. Nollet concluded that the pig bladder membrane was more
permeable to water than to alcohol. After this pioneering work, nothing much
happened on this frontier for a long time.
Then in the middle of the 19th century, a Berlin tradesman, Moritz Traube
enlivened this field by inventing the first man-made semi-permeable membrane
Page 27 of 43
67
. He did that by filling the open end of a glass tube with a solution of
potassium ferrocyanide. He then dipped the filled end of the tube into a solution
of copper sulfate. A reddish brown precipitate formed at the interface in the
shape of a thin membrane of a finite thickness, because once formed the
membrane is impermeable to both copper and ferrocyanide ions. To explain this
phenomenon, Traube introduced what he called “Atomic Sieve Theory”. That
is, the membrane has pores but they are so small that only water molecules can
pass through but not the larger copper and ferrocyande ions. Unfortunately this
artificial membrane was too fragile to handle and to conduct experimental
studies on. At this juncture, Wilhelm Pfeffer, a chemist with strong interest in
botany, came to the rescue 68;69.
Pfeffer made the copper ferrocyanide barrier to form within the
interstices of an unglazed porcelain pot by placing one of the two solutions in
the pot and by immersing the pot in the other solution. Now, the artificial
semipermeable membrane has strong mechanical support and can be handled
safely.
Pfeffer introduced a concentrated sucrose solution inside the pot and
connected the open upper end of the pot to a manometer. When he placed the
filled pot in a bucket of pure water, that water began to seep through slowly into
the pot. However, if a positive pressure of the right magnitude is applied to the
inside of the pot, it could completely stop the water flow. That pressure was
equal in magnitude to the osmotic pressure (π) of the concentrated sucrose
solution within the pot. Pfeffer’s highly accurate measurements demonstrated
that the osmotic pressure, π, is directly related to the concentration of sucrose
inside the pot and inversely related to the absolute temperature.
When the Dutch investigator, Hugo deVries 70, heard about these
outstanding achievements, he was all excited and passed the information to his
young Dutch fellow-scientist, J. H. van’t Hoff 71;72;73. It did not take too long for
van’t Hoff to reach the conclusion that the osmotic pressure can be
quantitatively described by the following equation:
π V = R’ T,
(5)
where V represents the volume of the solution containing one mole of sucrose.
R’ is a constant. From the data provided by Pfeffer, van’t Hoff showed that this
constant is very close to the gas constant, 1.987 cal/degree.
Page 28 of 43
The success of the Pfeffer’s model stimulated intensive effort further to
improve the instrument. Thus, in the hand of Morse 74, an osmotic pressure
created by a sucrose solution he studied lasted 60 days 74p66. In a parallel study
by de Vries himself, the protoplast of the root hair of red beets stayed shrunken
in a concentrated NaCl solution for as long as 7 days 75. Understandably, these
landmark records were once highly treasured and taken as convincing evidence
that van’t Hoff’s semipermeability indeed demonstrates the existence of two
kinds of substances. Some can go through the semipermeable membrane; others
cannot go through the semipermeable membrane. When they cannot go
through, they stay so not just for now, or for a few days, weeks, months or even
years but forever.
In the new perspective provided by the association-induction hypothesis,
these macroscopic notions ought to be replaced by their corresponding realistic
microscopic models. For example, as far back as 1909, Bigelow and Bartell 76
had already shown that unglazed porcelain pots without copper ferrocyanide or
other additives also produce an osmotic pressure and yet the pores could
measure 0.37 micron (3700 Ǻ) in diameter and thus two orders of magnitude
wider than the diameter of a “impermeant” sucrose molecule (9 Ǻ ). In term of
the microscopic model of the AI Hypothesis, this is a different kind of
phenomena from the macroscopic membrane permeability problem discussed
above. These gigantic pores were not empty cavities but space filled with
polarized-oriented multilayers of water molecules. This dynamically structured
water offers strong resistance to the passage of large molecules like sucrose---as
we have clearly demonstrated in the case of inverted frog skin in the earlier
section on Permeability. We could quantitatively measure the extremely low
permeability because we had the powerful tool of radioactive tracer technology.
But even molecules many times bigger than sucrose is not impermeable to the
best of Pfeffer’s modification of Traube’s “semipermeable” membrane. In
general principle, at above absolute zero temperature, the impermeable will all
permeate through sooner or later.
In contrast, we have more than just speculation to explain de Vries’s 7
day long maintained shrunken protoplast in concentrated NaCl solution of the
red beet root cell. Nasonov & Aizenberg 77 and by Kamnev 78 showed how in
the 1930’s.
Curve A in Figure 14
shows the time course of
shrinkage of isolated frog
muscles in a Ringer solution
Page 29 of 43
Figure 14
containing 4% sucrose. However, the cell membrane of frog muscle turned out
to be , quite permeable to sucrose as indicated by its accumulation in the cells
(curve B.)
In fact, to explain swelling as well as shrinkage in living cells in various
permeant solutes like sucrose, the AI Hypothesis offered a new theory. Its
testing and confirmation were conducted on many non-living “extrovert”
models 10p107like gelatin, oxygen-carrying linear polymers like polyethylene
glycol (PEG) polyethylene oxide (PEO), acid or alkali denatured proteins--models that cause the polarization-orientation of water molecules but not in
“introvert” models10p107 including all so-called “native proteins” 79.
Figure 15 shows
an example 79;10p256.
Enclosed in ¼ inch
dialysis tubing with
both ends tied, the
model expanded or
shrank in volume in a
lasting manner when
incubated in dilute and
concentrated solutions
of sodium citrate, to
which the dialysis
tubing
is
fully
permeable.
Figure 15
At about the same time the Russian scientists did their above-quoted
study on muscle shrinkage, the English protein chemist, Dorothy Jordan-Lloyd
of the Cambridge University measured the degree of swelling of living frog
muscle as a function of pH 80;5p248. Two peaks of extensive swelling were seen.
The rising limbs of each peak have a midpoint equal to the pK’s of major fixed
cations and anions respectively. As a part of the AI hypothesis, I suggested a
simple explanation: In normal frog muscle cells, salt linkages formed between
fixed cations and anions help to keep the steady volume of the muscle cells.
When the pH of the bathing medium approached the pK’s of the fixed anions
and fixed cations, the salt-linkages began to dissociate, thereby exposing the
polypeptide chains, on which more water becomes adsorbed and swelling
occurs as a result.
Page 30 of 43
But that was how far I could go in 1962. With the introduction of the
subsidiary polarized (oriented) multilayer theory of cell and cell model water in
1965, a new concept emerged: That is, if not restrained by salt linkages, the
polarized oriented multi-layers of water molecules would grow much deeper
and create more extensive swelling. But H+ and OH- are not the only external
mono-valent ions that at high enough concentrations can cause swelling in frog
muscle. Some familiar alkali-metal ions can also do the same.
It is well known that in an isotonic or 120 mM NaCl solution , frog
muscle cells can maintain their normal volume indefinitely. Yet von Korosy 81
and Martin Fischer 82 had repeatedly demonstrated extensive swelling of
isolated frog muscle in an isotonic KCl solution. In trying to explain this
phenomenon, my associates and I pointed out that the osmotic or vapor
pressures of these salt solutions at the same molarity are virtually identical. Nor
had the frog muscle’s unusual sensitivity to KCl anything to do with the intact
cell membrane barrier. For muscle fibers cut into 2 mm and 4 mm open-ended
segments that do not regenerate new cell membranes continue to respond to
isotonic KCl solution by extensive swelling. It was then pointed out that in
healthy animals, a normal amount of the EWC, ATP maintains the nanoprotoplasm in its resting living state. In this state, the β-, and γ-carboxyl groups
are maintained at their respective low c-value state, at which K+ is the
overwhelmingly preferred over Na+. As a result, the muscle cells remain
indifferent to the impact of isotonic NaCl in their blood and tissue fluids but
swell promptly on exposure to isotonic KCl.
Isotonic KCl is not something you find in your immediate surroundings.
Isotonic NaCl, on the other hand, is part of the milieu that keeps all your cells
alive. It is the major constituent of your body fluids or blood plasma. But it is
harmless under normal conditions.
However, every child can probably tell you how a fall or some other
mishap could cause a part of your body to undergo painful swelling. Physicians
and pathologists can also describe how a major auto accident, say, can cause a
concussion and life-threatening brain swelling. Once more the AI Hypothesis
sees a theoretical cause for the injury-induced tissue swelling of one sort or
another.
The critical change is that injury destroys or interrupts the local energy
metabolism to replace the ATP lost through injury. Being a most powerful
EWC, the failure of its replacement means that the c-value of the
β-, and γ-carboxyl groups will become higher sharply in consequence. Figure 3
cited on page 9 shows that such a sharp rise of c-value, the normal strong
preference of the β-, and γ-carboxyl groups for K+ gave way to strong
preference for Na+. Suddenly, the normally harmless, indeed vitally-needed
Page 31 of 43
near isotonic level of Na+ becomes as deadly as isotonic KCl to normal body
tissues. The corresponding breakup of shape and size-maintaining salt linkages
by the Na+ (and Cl- ions) caused tissue swelling.
To put this theory to an experimental
testing, Ling and Kwon kept isolated
mouse kidneys, brain and other organs in a
cold room maintained at 4oC in a oxygenpoor modified mammalian Ringer’s
solution 83. Each solution contained a
mixtures of salt and sucrose so that anyone
mixture is isotonic (see abscissa of Figure
16.) After some time, the tissues were
weighed and their water contents
determined. The results show that the final
weight
over
initial
weight
ratio
(Wfinal/Winitial) of the kidney tissue rose
sharply in NaCl and LiCl solutions of
increasing concentration. To produce
Figure 16
maximum swelling, both the specific cation
Na+ (or Li+) and Cl anion are essential. In solutions containing only sucrose and
MgSO4, no swelling occurred at all.
Figure
17
plots
the
concentration of ATP determined in
isolated brain tissue after different
length of time in the cold and
anoxia (time indicated in number of
hour near each data point in a
similar cold and anoxic isotonic
NaCl.) The data demonstrated
increasing swelling with decreasing
ATP contents. The maximum
Figure 17
swelling and hence highest water
content was found in brain tissue containing the least amount of ATP after the
longest period of cold and anoxic exposure.
Together, the data given in the last two figures confirm the theoretical
interpretation of the main mechanism of injury-induced tissue swelling: ATP
depletion raised the c-value of the β-, and γ-carboxyl groups and as a result
Page 32 of 43
Na+ became a strongly preferred cation. Since the tissue fluid and blood plasma
all contain a high concentration of NaCl, extensive tissue swelling is the
consequence. On this note all my past presentations of this story ended. I did
not notice then that there is a hidden question that needs to be answered. And
now the hidden question will be revealed and answered.
To do so, requires the invocation of the “Principle of Additivity”, which I
introduced in my first book published in 1962 5p95, including experimental
evidence supporting the Principle then available 5pp176to179. Best of all, the very
recently published work of Ling and Ochsenfeld 12 provided additional clear-cut
confirmation. In the shortest way of saying it, everything adsorbed on the
protein of a nano-protoplasm unit exercises an influence on its electronic
profile, though the polarity and magnitude varies from one adsorbent to another.
As the reader already knows, the electron density of each polypeptide
carbonyl oxygen atom is expressed in terms of its c-value analogue. For
partners, the carbonyl group has two alternative choices. On one hand, at high
c-value analogue, it prefers other peptide imino groups to form α-helical H
bonds. On the other hand, at low c-value analogue, the carbonyl groups prefer
to polarize-orient multilayers of water molecules. With the fall of ATP
concentration, the c-value of β-, and γ-carboxyl groups all rise higher, causing
the displacement of the fixed cations engaged in form- and size maintaining salt
linkages by Na+. Uptake of more water and cell swelling follow. The hidden
question now raised is: Would this electron-donating activity due to the fall of
ATP level also cause an enhancement of the high c-value analogues of the
carbonyl oxygen atoms and thus strengthen these form- and size-maintaining
salt linkages? The answer is no--- due to a strategic exercise of the “Principle
of Additivity”.
This Principle tells us that every single positively charged Na+ adsorbed
on a β-, or γ-carboxyl group is itself an electron-withdrawing agent. Together,
the electron-withdrawing influence of the multitude of adsorbed Na+ combine to
reverse the initial electron-donating action caused by the lost ATP to produce a
net electron withdrawing impact on all the form- and size-maintaining carbonyl
groups. At the low c-value analogue, all the backbone carbonyl oxygen atoms
shift to binding multiple layers of water molecules and swelling follows in
consequence
There are two mutually dependent advantages that make this transition
inevitable: numerical superiority of the number of effectors and much closer
proximity to the target groups.
From the work on cell swelling that Ling and Peterson published in 1966,
we discovered that alkali-chloride at high concentration could liberate between
100 to 200 mmoles of β-, and γ-carboxyl groups per kilogram of fresh muscle
Page 33 of 43
cells from the volume-and-shape maintaining salt linkages. In comparison, each
kilogram of fresh muscle contains only 5 mmoles of ATP. This numerical
superiority of the effector Na+ offers the first overwhelming advantage for the
electron withdrawing effect.
The second overwhelming advantage is proximity to the target groups.
Adsorbed on a high c-value sites, the Na+ is primarily in what is called
Configuration 0 5fig4p61. As such, the Na+ is fully dehydrated and thus in its small
naked form. Furthermore, the β-, and γ-carboxyl groups are carried on their
respective short side chains. Together, the positive charge at the center of each
adsorbed sodium ion is very close physically to the local peptide carbonyl
groups, In contrast, there is only 5 mM of cardinal sites for ATP. Their electron
donating influence is on the average far away from the backbone carbonyl
oxygen atoms under their influence. The overall result is that the adsorbed Na+
strongly lower their c-value analogue, cause the dissociation of the form- and
size-maintaining salt linkages and bring about extensive water polarization and
orientation in multi-layers. And injury induced tissue-swelling follows as we
know well from experience.
Page 34 of 43
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